The field of the present invention relates to tissue barrier assays for screening formulations and chemical compositions.
In vitro analysis of the movement of compounds (e.g., drugs) across an epithelial barrier, such as intestinal epithelium or airway epithelium, is typically performed using an Ussing-type chamber. To perform a tissue barrier assay using an Ussing-type chamber, a piece tissue is removed as an intact sheet from the body and mounted in a device which contains an enclosed, internal hollow chamber such that it divides the internal chamber into two separate chambers. Thereafter, biologically compatible solutions are filled into both chambers, and the drug of interest is added to one chamber""s solution. Samples are then removed from the contralateral chamber solution at various times to determine the rate at which the drug moves across the tissue barrier. This type of tissue barrier assay is cumbersome, inefficient, and only permits a very limited number of independent samples to be derived from a unit area of tissue sheet.
Transdermal delivery of drugs is a type of tissue transfer that involves transfer of the drug from a transdermal drug delivery device through the skin and into the patient""s blood stream. Transdermal drug delivery offers many advantages compared to other methods of drug delivery. One obvious advantage is that needles and the associated pain are avoided. This is especially desirable for drugs that are repeatedly administered. Avoiding the unpleasantness of needles would also lead to improved patient compliance of drug regimens.
Another advantage of transdermal drug delivery is its ability to offer prolonged or sustained delivery, potentially over several days to weeks. Other delivery methods, such as oral or pulmonary delivery, typically require that the drug be given repeatedly to sustain the proper concentration of drug within the body. With sustained transdermal delivery, dose maintenance is performed automatically over a long period of time. This is especially beneficial for drugs with short half-lives in the body, such as peptides or proteins.
A final advantage is that drug molecules only have to cross the skin to reach the bloodstream when given transdermally. Transdermally administered drugs bypass first-pass metabolism in the liver, and also avoid other degradation pathways such as the low pH""s and enzymes present in the gastrointestinal tract.
The skin is the largest organ of the body. It is highly impermeable to prevent loss of water and electrolytes. It is subdivided into two main layers: the outer epidermis and the inner dermis. The epidermis is the outer layer of the skin, 50 to 100 micron thick (Monteiro-Riviere, 1991; Champion, et al., 1992). The dermis is the inner layer of the skin and varies from 1 to 3 mm in thickness. The goal of transdermal drug delivery is to get the drug to this layer of the skin, where the blood capillaries are located, to allow the drug to be systemically delivered. The epidermis does not contain nerve endings or blood vessels. The main purpose of the epidermis is to generate a tough layer of dead cells on the surface of the skin, thereby protecting the body from the environment. This outermost layer of epidermis is called the stratum corneum, and the dead cells that comprise it are called corneocytes or keratinocytes.
The stratum corneum is commonly modeled or described as a brick wall (Elias, 1983; Elite, 1988). The xe2x80x9cbricksxe2x80x9d are the flattened, dead corneocytes. Typically, there are about 10 to 15 corneocytes stacked vertically across the stratum corneum (Monteiro-Riviere, 1991; Champion et al., 1992). The corneocytes are encased in sheets of lipid bilayers (the xe2x80x9cmortarxe2x80x9d). The lipid bilayer sheets are separated by xcx9c50 nm. Typically, there are about 4 to 8 lipid bilayers between each pair of corneocytes. The lipid matrix is primarily composed of ceramides, sphingolipids, cholesterol, fatty acids, and sterols, with very little water present (Lampe et al., 1983 [a]; Lampe et al., 1983 [b]; Elias, 1988).
Although it is the thinnest layer of the skin, the stratum corneum is the primary barrier to the entry of molecules or microorganisms across the skin. Most molecules pass through the stratum corneum only with great difficulty, which is why the transdermal drug delivery route has not been more widely used to date. Once the molecules have crossed the stratum corneum, diffusion across the epidermis and dermis to the blood vessels occurs rapidly. Thus, most of the attention in transdermal drug delivery research has been focused on transporting molecules and drugs across the stratum corneum.
The most common form of transdermal drug delivery device is the transdermal drug xe2x80x9cpatch,xe2x80x9d where a drug, or pharmaceutical, is contained within a reservoir placed next to the skin (Schaefer and Redelmeier, 1996). The drug molecules typically cross the skin by simple diffusion. Transport is governed by the rate of molecular diffusion into and out of the skin, and partitioning of the drug into the skin. Generally speaking, transdermal drug delivery is limited to small, lipophilic molecules such as scopolamine, nitroglycerine, and nicotine, which readily permeate the skin. The delivery is slow, typically taking hours for the drug to cross the skin, and treatment is only effective when a very small amount of drug is required to have a biological effect (Guy and Hadgraft, 1989).
Since transdermal delivery can be slow, many substances have been used to enhance molecular transport rates. These substances are known as chemical enhancers or penetration enhancers. Chemical enhancers increase the flux of a drug through the skin by increasing the solubility of drug in the stratum corneum or increasing the permeability of drug in the stratum corneum. There are many possible enhancers and the selection is further complicated by the fact that combinations of enhancers are known to improve drug flux beyond what would be expect due to the presence of each constituent independently.
Transdermal drug delivery devices, such as a transdermal patch, also generally contain an adhesive, which serves to keep the device in intimate contact with the skin, and may also form the matrix in which the drug is dissolved or dispersed. There are many different forms of adhesives that can be used, and it is often a very difficult problem to select which adhesive to use with any drug or drug and enhancer.
Currently, the choice of appropriate adhesive and enhancers and their relative proportion with respect to the drug is only determined by general guidelines from what is known to be safe and what may have been effective with other drugs. The vast majority of the formulation development is made through trial and error experimentation.
Most transdermal transport experiments to date have utilized a relatively large human skin diffusion cell in which a source side includes a drug solution with additives and a sink side that typically includes saline solution or some other solution that is thought to model the dermis. The skin membrane separates the two sides of the cell, and is most often stratum corneum cadaver skin that has been carefully separated from the whole skin sample supplied by a tissue bank. The volume of the device is typically 5 cc or greater. Samples are periodically taken from the sink side of the cell to determine the flux of drug through the stratum corneum film. The entire procedure is very laborious and requires the use of large quantities of skin, which is extremely difficult to obtain. Therefore, only a relatively small number of the many possible combinations of chemical entities can be examined.
Thus, there remains a need in the art for a method for screening a large number samples to identify optimal compositions or formulations for tissue barrier transport, including transdermal transport, of compounds, pharmaceuticals and other components.
The present invention relates to high-throughput systems and methods to prepare a large number of component combinations, at varying concentrations and identities, at the same time, and high-throughput methods to test tissue barrier transfer of components in each combination. The methods of the present invention allow determination of the effects of additional or inactive components, such as excipients, carriers, enhancers, adhesives, and additives, on transfer of active components, such as pharmaceuticals, across tissue, such as skin, lung tissue, tracheal tissue, nasal tissue, bladder tissue, placenta, vaginal tissue, rectal tissue, stomach tissue, gastrointestinal tissue, and eye or corneal tissue. The invention thus encompasses the high-throughput testing of pharmaceutical compositions or formulations in order to determine the overall optimal composition or formulation for improved tissue transport, including without limitation, transdermal transport. Specific embodiments of this invention are described in detail below.
In one embodiment, the invention concerns an apparatus for measuring transfer of components across a tissue, comprising a support plate, an array of samples supported by the support plate, a membrane or tissue specimen overlaying the array of samples, and a reservoir plate secured to a side of the membrane or tissue specimen opposite the array of samples. In one aspect of the invention, each sample in the array contains a unique composition or formulation of components, wherein different active components or different physical states of an active component are present in one or more of the samples in the sample array.
In another aspect of the present invention, each sample of the array includes a component-in-common and at least one additional component, wherein each sample differs from at least one other sample with respect to at least one of:
(i) the identity of the additional components,
(ii) the ratio of the component-in-common to the additional components, or
(iii) the physical state of the component-in-common.
A xe2x80x9ccomponent-in-commonxe2x80x9d is a component that is present in every sample in a sample array. In one embodiment, the component-in-common is an active component, and preferably, the active component is a pharmaceutical, dietary supplement, alternative medicine or a nutraceutical. The samples may be in the form of liquids, solutions, suspensions, emulsions, solids, semi-solids, gels, foams, pastes, ointments, or triturates.
In another embodiment, the invention concerns a method of measuring tissue barrier transport of a sample, comprising:
(a) preparing an array of samples having an active component and at least one additional component, wherein each sample differs from at least one other sample with respect to at least one of:
(i) the identity of the active component;
(ii) the identity of the additional components,
(iii) the ratio of the active component to the additional components, or
(iv) the physical state of the active component;
(b) overlaying the array of samples with a tissue specimen;
(c) securing a reservoir plate to a side of the tissue specimen opposite the array of samples, the plate having an array of reservoirs corresponding to the array of samples;
(d) filling the array of reservoirs with a reservoir medium; and
(e) measuring concentration of the active component in each reservoir at one or more time points to determine transport of the active component from each sample across the tissue specimen.
In a preferred embodiment, the active component is a pharmaceutical, a dietary supplement, an alternative medicine, or a nutraceutical. In another embodiment, the tissue specimen is skin.
In another embodiment, the invention concerns a method of analyzing or measuring flux of a sample across a tissue, comprising:
(a) preparing an array of samples having a component-in-common and at least one additional component, wherein each sample differs from at least one other sample with respect to at least one of:
(i) the identity of an active component;
(ii) the identity of the additional components,
(iii) the ratio of the component-in-common to the additional components, or
(iv) the physical state of the component-in-common;
(b) overlaying the array of samples with a tissue specimen;
(c) securing a reservoir plate to a side of the tissue specimen opposite the array of samples, the plate having an array of reservoirs corresponding to the array of samples;
(d) filling the array of reservoirs with a reservoir medium; and
(e) measuring concentration of the component-in-common in each reservoir as a function of time to determine flux of the component-in-common from each sample across the tissue specimen.
In an alternative embodiment, the method comprises an additional step of cutting the tissue specimen to avoid lateral diffusion between wells. The method preferably comprises analyzing the tissue specimen for defects, or inhomogeneities, and correcting for or repairing the defects.
In another embodiment, the invention concerns an apparatus and method of high-throughput screening of active component or drug flux through the stratum corneum recognizing that such flux is determined, at least in part, by the permeability of the drug within the tissue in the presence of an enhancer. The permeability is generally governed by at least two factors: the solubility of the active component or drug within the stratum corneum and the diffusivity of the active component or drug within the stratum corneum. These two factors, solubility and diffusivity, can be measured independently as a method of indirectly assessing the flux through the stratum corneum. Thus, an array of wells containing samples of different compositions of active component and inactive compounds, including without limitation, compositions comprising active component/carrier or excipient/, active component/carrier or excipient/enhancer/, active component/adhesive/enhancer/additive, are constructed. Known amounts of stratum corneum are added to each well and the rate at which the active component or drug is taken up into the tissue sample is measured by extracting the tissue from similarly prepared wells at different times. Measuring the concentration after times sufficiently long so that the amount dissolved is not changing with time can assess the equilibrium concentration of active component or drug within the tissue. The product of the rate and solubility is proportional to the permeability of the active component or drug.
The high-throughput combinatorial screening systems and methods of the present invention identify optimal compositions or formulations to achieve a desired result for such compositions or formulations, including without limitation, construction of a transdermal delivery device. In particular, the systems and methods of the present invention may be used to identify 1) optimal compositions or formulations comprising one or more active components and one or more inactive components for achieving desired characteristics for such compositions or formulations, 2) optimal adhesive/enhancer/additive compositions for compatibility with a drug, 3) optimal drug/adhesive/enhancer/additive compositions for maximum drug flux through stratum corneum, and 4) optimal drug/adhesive/enhancer/additive composition to minimize cytotoxicity